Methotrexate (MTX) is one of the most effective drugs for RA, but 20- 30% of patients have suboptimal clinical responses to MTX, and 15-25% have side effects limiting its use. Thus, it is important to elucidate influences on MTX efficacy and toxicity. We will test the hypothesis that single nucleotide polymorphisms (SNPs) in genes encoding key enzymes involved in folate or MTX metabolism or in the mechanism of actions of MTX (e.g. the adenosine pathway) influence clinical responses to MTX. We are uniquely positioned to utilize clinical outcomes (ACR response criteria, radiographic progression and toxicities) and genomic DNA from patients in two completed clinical trials: 153 MTX-treated RA patients from an Immunex trial comparing MTX and etanercept, and 79 MTX-treated RA patients from a UAB trial of folic acid supplementation. HLA DRB1 alleles and a total of 5 known SNPs in the following 4 key genes will be genotyped: 1) 5,10- methylenetetrahydrofolate reductase (MTHFR); 2) 5-methyl- tetrahydrofolate-homocysteine methyltransferase (methionine synthase) (MTR); 3) methionine synthase reductase (MTRR); and 4) adenosine receptor A2A [A(2A)R]. These SNPs were chosen on the basis of being common enough in the general population to allow meaningful analyses, their key roles in relevant pathways, and evidence of their biological activity. Through the MCRC Methodology Core, we will look for associations between SNP alleles and MTX efficacy or toxicity. Although these known SNPs are important, SNP haplotypes may be even more informative, aqs they allow characterization of the effect of multiple SNPs working in concert. Therefore, we will use both "in silico" in sequencing approaches to identify novel SNP haplotypes in these 4 and 3 other critical genes: dihydrofolate reductase (DHFR), 5- aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR- T), and aldehyde oxidase (AO). In addition to data mining of public domain and proprietary (i.e. Celera) SNP databases, we will perform SNP discovery on 40 individuals from two racial/ethnic groups [20 African-American (A-A) and 20 Caucasian]. Differences in frequencies of novel haplotype related to disease status or race/ethnicity will be sought by analysis of 108-AA Ra patients and 53-AA controls; 336 RA patients (mostly Caucasian); and 800 controls (mostly Caucasian) from established cohorts. Based on results from these studies, the role of selected novel SNP haplotypes on MTX efficacy and toxicity will be tested in patients from the folic acid and Immunex trials. We will compare the predictive power of two approaches to genetic profiling: the single SNP approach and the SNP haplotype approach. These studies may provide clinically useful markers of MTX efficacy or toxicity in RA.